BioMed Research International

Volume 2013 (2013), Article ID 460619, 9 pages

http://dx.doi.org/10.1155/2013/460619

Research Article

Synthesis and Biological Evaluation of O-[3-¹⁸F-fluoropropyl]-α-methyl Tyrosine in Mesothelioma-Bearing Rodents

I-Hong Shih, Fan-Lin Kong, Mohammad S. Ali, Yinhan Zhang, Dong-Fang Yu, Xudong Duan, and David J. Yang

Department of Experimental Diagnosis Imaging, Unit 59, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA

Received 12 April 2013; Accepted 6 June 2013

Academic Editor: Tomio Inoue

Copyright © 2013 I-Hong Shih et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Radiolabeled tyrosine analogs enter cancer cells via upregulated amino acid transporter system and have been shown to be superior to ¹⁸F-fluoro-2-deoxy-D-glucose (¹⁸F-FDG) in differential diagnosis in cancers. In this study, we synthesized O-[3-¹⁹F-fluoropropyl]-α-methyl tyrosine (¹⁹F-FPAMT) and used manual and automated methods to synthesize O-[3-¹⁸F-fluoropropyl]-α-methyl tyrosine (¹⁸F-FPAMT) in three steps: nucleophilic substitution, deprotection of butoxycarbonyl, and deesterification. Manual and automated synthesis methods produced ¹⁸F-FPAMT with a radiochemical purity >96%. The decay-corrected yield of ¹⁸F-FPAMT by manual synthesis was 34% at end-of-synthesis (88 min). The decay-corrected yield of ¹⁸F-FPAMT by automated synthesis was 15% at end-of-synthesis (110 min). ¹⁸F-FDG and ¹⁸F-FPAMT were used for in vitro and in vivo studies to evaluate the feasibility of ¹⁸F-FPAMT for imaging rat mesothelioma (IL-45). In vitro studies comparing ¹⁸F-FPAMT with ¹⁸F-FDG revealed that ¹⁸F-FDG had higher uptake than that of ¹⁸F-FPAMT, and the uptake ratio of ¹⁸F-FPAMT reached the plateau after being incubated for 60 min. Biodistribution studies revealed that the accumulation of ¹⁸F-FPAMT in the heart, lungs, thyroid, spleen, and brain was significantly lower than that of ¹⁸F-FDG. There was poor bone uptake in ¹⁸F-FPAMT for up to 3 hrs suggesting its in vivo stability. The imaging studies showed good visualization of tumors with ¹⁸F-FPAMT. Together, these results suggest that ¹⁸F-FPAMT can be successfully synthesized and has great potential in mesothelioma imaging.

1. Introduction

Numerous studies have demonstrated that growing cancer cells have higher metabolism of glucose and amino acids than other cells in the body. One well-known modality for imaging the metabolic activity of cancers is positron emission tomography (PET) using ¹⁸F-2-fluoro-2-deoxy-D-glucose (¹⁸F-FDG), the current gold standard for cancer diagnosis [1]. However, ¹⁸F-FDG has limitations such as poor differentiation between low-grade tumor and normal tissues in brain [2] and between tumor and inflamed or infected tissues [3]. Radiolabeled amino acids offer higher specificity in characterizing tumors than ¹⁸F-FDG does. In particular, radiolabeled aromatic amino acids are attractive alternatives to ¹⁸F-FDG because of easier chemistry alteration and their ability of detection of upregulated amino acid transporters [4], which indirectly reveal cell proliferation. Therefore, ¹¹C- and ¹⁸F-labeled amino acid analogs were developed as alternative metabolic imaging tracers for PET.

¹¹C-methyl methionine (¹¹C-MET) and L-1-¹¹C tyrosine (¹¹C-TYR) have been commonly used for clinical research and practices. Unfortunately, the half-life of ¹¹C is only 20 min, and, therefore, ¹¹C-labeled amino acid analogs require an inconvenient on-site synthesis which reduces their broad clinical usages. ¹⁸F has a half-life of 110 min, and it can be used at a centralized remote facility to synthesize radiolabeled compounds which can then be delivered to different hospitals simultaneously. Moreover, low β ⁺-energy of ¹⁸F causes a short positron linear range in tissue, thereby providing high resolution in PET images. A number of ¹⁸F-labeled amino acid analogs in PET have been investigated, including L-2-¹⁸F-fluorotyrosine (¹⁸F-TYR) [5], O-2-¹⁸F-fluoroethyl-L-tyrosine (¹⁸F-FET) [6], and L-3-¹⁸F-fluoro-α-methyl tyrosine (¹⁸F-FAMT). Recently, Wiriyasermkul et al. found that, unlike ¹⁸F-TYR, ¹⁸F-FET, and other ¹⁸F-labeled amino acids, ¹⁸F-FAMT is transported into cells through L-type amino transporter 1, which contributes to its highly tumor-specific accumulation [4]. ¹⁸F-FAMT was first studied as a brain-imaging probe [7]; later, its use in detecting oral squamous cell carcinoma [8], nonsmall cell lung cancer [9], and esophageal squamous cell carcinoma [10] was investigated. However, the yield of ¹⁸F-labeled amino acids by an electrophilic fluorination reaction is low (17% for ¹⁸F-TYR [5]; 20% ± 5.1% for ¹⁸F-FAMT [11]). Wester et al. synthesized O-2-¹⁸F-fluoroethyl-L-tyrosine (¹⁸F-FET) by a nucleophilic fluorination reaction in about 50 min with an overall radiochemical yield of 40% and evaluated it as a PET tracer for cerebral and peripheral tumors [6]. Hamacher and Coenen synthesized ¹⁸F-FET using one-pot reaction, and the radiochemical yield obtained within 80 min was about 60% [12]. However, both methods require high-performance liquid chromatography (HPLC) for purification, which limits the possibility of automated synthesis. Wang et al. obtained ¹⁸F-FET by direct nucleophilic fluorination reaction of the protected precursor N-butoxycarbonyl-(O-(2-tosyloxyethyl))-L-tyrosine methyl ester, followed by a rapid removal of the protecting group, and a labeled intermediate was separated out with Sep-Pak silica plus cartridge [13]. The radiochemical yield was about 40% at the end of synthesis (50 min). Bourdier et al. used this method for automated radiosynthesis of ¹⁸F-FET, and the yield was about 35% within 63 min [14]. ¹⁸F-FET was widely used in clinical studies in patients with high-grade or low-grade glioma [15, 16].

Despite the very promising clinical results of ¹⁸F-FAMT, existing methods for synthesizing ¹⁸F-FAMT produce a low chemical yield, which limits the availability of the compound for clinical use, and they require high-performance liquid chromatography (HPLC) for purification, which precludes the use of an automated module to synthesize ¹⁸F-FAMT. Therefore, it is desirable to develop an ¹⁸F-FAMT analog with high chemical yield that can be applied clinically in most major medical facilities. In the present study, we synthesized unlabeled O-[3-¹⁹F-fluoropropyl]-α-methyl tyrosine (¹⁹F-FPAMT) and ¹⁸F-labeled O-[3-¹⁸F-fluoropropyl]-α-methyl tyrosine (¹⁸F-FPAMT) by using nucleophilic substitution to place a fluorine atom on the aliphatic chain of α-methyl tyrosine and solid-phase extraction (SPE) column to purify the products. We then used our customized, fully automated synthesis module to synthesize ¹⁸F-FPAMT. Finally, we used a rat mesothelioma model to investigate the feasibility of using ¹⁸F-FPAMT as a tumor-seeking imaging agent.

2. Materials and Methods

2.1. General

All chemicals and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA). Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker 300 MHz Spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA), and mass spectra were recorded on a Waters Q-TOF Ultima mass spectrometer (Waters, Milford, MA, USA) at the Chemistry Core Facility at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). An HPLC system (Waters) was integrated with an ultraviolet detector and a flow-count radio-HPLC detector (BioScan Inc., Washington, DC, USA). The analyses of radio-thin layer chromatography (TLC) were performed on radio-TLC Imaging Scanner (BioScan, Inc.). The scintigraphic imaging studies were processed on microPET (Siemens Medical Systems, Inc., Malvern, PA, USA).

2.2. Synthesis of N-t-butoxycarbonyl-O-[3-tosylpropyl]-α-methyl Tyrosine Ethyl Ester

N-t-butoxycarbonyl-O-[3-hydroxypropyl]-α-methyl tyrosine ethyl ester, which we used as the precursor compound for synthesis of ¹⁹F-FPAMT and ¹⁸F-FPAMT, was prepared as described previously [17]. Briefly, N-t-butoxycarbonyl-O-[3-hydroxypropyl]-α-methyl tyrosine ethyl ester (490 mg; 1.28 mmol) in anhydrous pyridine (32 mL) was cooled to 0°C. Paratoluenesulfonyl chloride (1015 mg; 5.32 mmol) was added to this solution, and the solution was stirred for 30 min. The reaction mixture was then stored in a refrigerator overnight. The mixture was filtered, and the filtrate was poured into an ice and water mixture and extracted with diethyl ether. The ethereal solvent was washed with 30 mL of hydrochloric acid and water (1 : 1, v/v) to remove pyridine, and the solvent was dried over anhydrous MgSO₄. After filtration and solvent evaporation, N-t-butoxycarbonyl-O-[3-tosylpropyl]-α-methyl tyrosine ethyl ester was purified by column chromatography using a silica gel column and eluted with hexane and ethyl acetate (2 : 1, v/v) to yield 430 mg (62.5%). NMR and mass spectrometry were performed to confirm the structures.

2.3. Synthesis of   ¹⁹F-FPAMT

We used a three-step procedure to synthesize ¹⁹F-FPAMT (Figure 1). The first step was a displacement reaction. Kryptofix 222 (253.9 mg; 0.67 mmol) and K¹⁹F (40.5 mg; 0.69 mmol) were added to a vial containing N-t-butoxycarbonyl-O-[3-tosylpropyl]-α-methyl tyrosine ethyl ester (compound 1; 390 mg; 0.75 mmol) in acetonitrile (1 mL). The reaction vial was heated under reflux at 90°C for 40 min. After heating, the solution was evaporated to dryness. The mixture was reconstituted in 0.5 mL of ethyl acetate. N-t-butoxycarbonyl-O-[3-¹⁹F-fluoropropyl]-α-methyl tyrosine ethyl ester (compound 2) was purified by column chromatography using a silica gel column and eluted with hexane and ethyl acetate (4 : 1, v/v) to yield 120.0 mg of the compound. The second step was to deprotect butoxycarbonyl (BOC), and the third step was to remove ethyl ester groups. O-[3-¹⁹F-fluoropropyl]-α-methyl tyrosine ethyl ester (compound 3) was synthesized by reacting N-t-butoxycarbonyl-O-[3-¹⁹F-fluoropropyl]-α-methyl tyrosine ethyl ester (compound 2; 82.3 mg; 0.30 mmol) with trifluoroacetate (0.7 mL) in dichloromethane (2.0 mL) at room temperature for 50 min. After the solvent was evaporated to dryness, sodium hydroxide (1 N; 1.0 mL) in methanol (1.0 mL) was added, and the mixture was heated at 90°C for 15 min to remove ethyl ester group. The mixture was passed through a 0.22 μM filter to yield ¹⁹F-FPAMT (compound 4). NMR and mass spectrometry were used to confirm the structure of this compound.

Figure 1: Synthetic scheme of FPAMT. The KF and kryptofix complex were incubated with N-t-butoxycarbonyl-O-[3-tosylpropyl]-α-methyl tyrosine ethyl ester (compound 1) in acetonitrile for synthesis of N-t-butoxycarbonyl-O-[3-¹⁹F-fluoropropyl]-α-methyl tyrosine ethyl ester (compound 2). After deprotection of butoxycarbonyl (BOC) of compound 2, O-[3-¹⁹F-fluoropropyl]-α-methyl tyrosine ethyl ester (compound 3) was synthesized. The final step is to yield FPAMT (compound 4) by deesterification of compound 3.

2.4. Manual Radiosynthesis of ¹⁸F-FPAMT

[¹⁸F]Fluoride in kryptofix complex (100 mCi in 0.3 mL acetonitrile) was purchased from the cyclotron facility of Cyclotope (Houston, TX, USA). N-t-butoxycarbonyl-O-[3-tosylpropyl]-α-methyl tyrosine ethyl ester (2 mg; 3.83 μmol) dissolved in acetonitrile (0.1 mL) was added to the [¹⁸F]fluoride-kryptofix complex (51.5 mCi). The reaction mixture was heated at 90°C for 15 min to allow the displacement to occur. After the reaction mixture cooled, it was passed through a 500 mg silica gel packed SPE column (Whatman Lab., Clifton, NJ, USA) and eluted with acetonitrile (2 mL). The acetonitrile was then evaporated in vacuo at 85°C. The resulting mixture was hydrolyzed with trifluoroacetate (0.2 mL) in dichloromethane (0.2 mL) at room temperature for 10 min to deprotect BOC. After the solvent was evaporated to dryness in vacuo, sodium hydroxide (1 N; 0.2 mL) in methanol (0.2 mL) was added and heated at 90°C for 15 min to remove ethyl ester group. After methanol evaporated, hydrochloric acid (0.1 N; 0.2 mL) was used to adjust the pH of the final product to 6.5. Radio-TLC and HPLC were performed to assure the purity and identity of the product.

2.5. Automated Radiosynthesis of ¹⁸F-FPAMT

The automated radiosynthesis of ¹⁸F-FPAMT was achieved by our customized automated module. The diagram of this automated module is shown in Figure 2. The automated radiosynthesis consisted of three steps: nucleophilic substitution, deprotection of BOC, and deesterification. Before radiosynthesis was completed, the reaction vial 1 (RV1) was preloaded with N-t-butoxycarbonyl-O-[3-tosylpropyl]-α-methyl tyrosine ethyl ester (6.2 mg; 11.8 μmol), and three syringes were loaded with different solutions: acetonitrile (3.0 mL), trifluoroacetate in dichloromethane (2.5 mL; 1 : 1, v/v), and sodium hydroxide in ethyl alcohol (1 N; 3.0 mL; 1 : 2, v/v). For the nucleophilic substitution, [¹⁸F]fluoride-kryptofix complex (0.2 mL; 29.36 mCi) was manually injected into the RV1 through the injection hole, and additional acetonitrile (0.35 mL) was manually injected into the RV1 to flush the residual [¹⁸F]fluoride-kryptofix complex inside the flow channel. Following this step, the infrared (IR) heater automatically heated the RV1 at 90°C for 15 min. For free fluoride separation, the mixture in the RV1 was automatically passed through a silica gel packed column (SPE 500 mg; Whatman Lab., Clifton, NJ, USA) to the reaction vial 2 (RV2) via nitrogen flow. Additional acetonitrile (2.0 mL) was then added to RV1, and the residual mixture was filtered through a SPE column to remove the free fluoride. The solution inside RV2 was evaporated in vacuo at 90°C for 15 min before deprotection of BOC was performed. Trifluoroacetate in dichloromethane (0.4 mL) was loaded into RV2, and the solution was set under room temperature for 10 min to allow the reaction to finish. The solvent was then evaporated to dryness in vacuo for 15 min. For deesterification, sodium hydroxide in methanol (0.6 mL) was loaded into RV2. The reaction mixture in RV2 was heated at 90°C for 15 min. Once deesterification was completed, the solvent in RV2 was evaporated in vacuo, and the radioactivities of the solvent in the column, RV1, and RV2 were measured upon the completion of ¹⁸F-FPAMT. Radio-TLC and HPLC were performed to assure the purity and identity of the final product.

Figure 2: Diagram of this customized automated synthesis device. Radioisotope can be manually injected into reaction vial 1 (RV1) through the manual injection port. The upper five green blocks are syringes which were loaded with different chemicals for synthesis or cleaning process. Other blue blocks are fixed parts, such as valves, vials, and columns.

2.6. In Vitro Cellular Uptake Studies

Rat mesothelioma IL-45 cells were maintained in the mixtures of Dulbecco's modification of Eagle's medium, F-12 (GIBCO, Grand Island, NY, USA), and 10% phosphate-buffered saline at 37°C in a humidified atmosphere containing 5% CO₂. Cells were plated onto 6-well tissue culture plates (2 × 10⁵ cells/well) and incubated with ¹⁸F-FPAMT (8 μCi/well) or ¹⁸F-FDG (Cyclotope, Houston, TX, USA; 8 μCi/well) for 0–2 h. After incubation, the cells were collected, and their radioactivity was measured using a gamma counter. Data were expressed as the mean percent ± the standard deviation of the cellular uptake of ¹⁸F-FPAMT or ¹⁸F-FDG.

2.7. Biodistribution of ¹⁸F-FPAMT and ¹⁸F-FDG in Mesothelioma-Bearing Rats

Three hundred forty-four female Fischer rats (140–185 g) were obtained from Harlan, Inc. (Indianapolis, IN, USA). The rats were housed in an animal facility at The University of Texas MD Anderson Cancer Center. All protocols involving animals were approved by the Animal Use and Care Committee at MD Anderson Cancer Center. Nine rats were inoculated with mesothelioma IL-45 cells (1 × 10⁵ cells/rat) at the hinged leg. Twelve days after being inoculated with the mesothelioma cells, the rats were anesthetized with ketamine (10–15 mg/rat). ¹⁸F-FPAMT dissolved in saline (0.5 mCi/5 mL) was injected intravenously into 9 rats ( rats/group, 30 μCi/rat,). For comparison, the clinical standard, ¹⁸F-FDG (Cyclotope,), was injected intravenously into 9 rats ( rats/group; 30 μCi/rat). The distribution of ¹⁸F-FPAMT or ¹⁸F-FDG in various tissues was assessed at 30 min, 1.5 hrs, and 3 hrs after injection by COBRA. Percent of injected dose per tissue type was then calculated, and the data were expressed as the mean percent ± the standard deviation of the injected dose.

2.8. Dosimetry of ¹⁸F-FPAMT and ¹⁸F-FDG

Dosimetric calculations were performed from 30 to 180 min after the administration of ¹⁸F-FPAMT and ¹⁸F-FDG, and time-activity curves were generated for each organ. Analytic integration of the curves was used to determine the area under the curve (AUC), which was divided by the injected dose to yield the residence times of ¹⁸F-FPAMT and ¹⁸F-FDG in each organ. Residence times were then used to calculate target organ absorbed radiation doses based on the medical internal radiation dosimetry methodology for the normal adult male using the Olinda software package (Oak Ridge, TN, USA).

2.9. PET Imaging of Mesothelioma-Bearing Rats

Mesothelioma-bearing rats cells were imaged when their tumors were 1-2 cm in diameter. The rats were anesthetized with 2% isoflurane and administered with 500 μCi of ¹⁸F-FDG or 500 μCi of ¹⁸F-FPAMT. Four serial 15-minute transaxial PET images of each rat were obtained using microPET (Siemens Medical Systems, Inc., IL, USA).

3. Results

3.1. Chemistry

The synthetic schemes of ¹⁸F-FPAMT and ¹⁹F-FPAMT are shown in Figure 1. The structure of precursor N-t-butoxycarbonyl-O-[3-tosylpropyl]-α-methyl tyrosine ethyl ester (compound 1) was confirmed using ¹H-NMR and mass spectrometry. The ¹H-NMR (CDCl₃) result was the following: (d, 2 H,  Hz), 7.26 (d, 2 H,  Hz), 6.97 (d, 2 H,  Hz), 6.67 (d, 2 H,  Hz), 4.23 (t, 2 H,  Hz), 4.12 (q, 2 H,  Hz,  Hz), 3.92 (t, 2H,  Hz), 3.22 (q, 2H,  Hz,  Hz), 2.40 (s, 3 H), 2.12 (m, 2 H), 1.54 (s, 3 H), 1.47 (s, 9 H), and 1.29 (t, 3 H,  Hz) ppm; M/Z: 558.29 (M+Na)⁺.

¹⁹F-FPAMT was obtained after subjecting compound 1 to nucleophilic substitution, free fluoride separation, deprotection of BOC, and deesterification. The structure of ¹⁹F-FPAMT (compound 4) was confirmed using ¹H-NMR and mass spectrometry. The ¹H-NMR (D₂O) result the following result was: (d, 2 H,  Hz), 6.93 (d, 2 H,  Hz), 4.75 (t, H,  Hz), 4.59 (t, H,  Hz), 4.13 (t, 2 H,  Hz), 2.84 (dd,  Hz,  Hz), 2.14 (m, 2 H), and 1.29 (s, 3 H) ppm. ¹⁹F-NMR ; M/Z: 406.38 (M+Na)⁺.

3.2. Radiochemistry

The ¹⁸F-displacement reaction produced 35.4 mCi (yield: 78%, decay corrected) of N-t-butoxycarbonyl-O-[3-¹⁸F-fluoropropyl]-α-methyl tyrosine ethyl ester, and the residual in the column was 3.77 mCi (8.3%, decay corrected). The no-carrier-added displacement product corresponded to the unlabeled N-t-butoxycarbonyl-O-[3-fluoropropyl]-α-methyl tyrosine ethyl ester under the same TLC system (hexane : ethyl acetate; 10 : 3, v/v) and HPLC system (20 μL loop, 210 nm, Bondapak CN-RP column, Waters, eluted with methanol : water, 3 : 2, v/v; flow rate 1.0 mL/min). The retention factor () of N-t-butoxycarbonyl-O-[3-¹⁸F-fluoropropyl]-α-methyl tyrosine ethyl ester was 0.46 with purity >99%. Under the same conditions, the value for [¹⁸F]fluoride in kryptofix complex was 0.1. After hydrolysis, ¹⁸F-FPAMT stayed at origin (). The retention times for N-BOC and the ethyl ester form of tosylpropyl-, fluoropropyl-, and ¹⁸F-fluoropropyl-α-ethyltyrosine were 16.13, 8.37, and 8.79 min, respectively. The decay-corrected yield for hydrolysis (deprotection of BOC and deesterification) was 89%. At the end-of-synthesis (88 min), 10 mCi of ¹⁸F-FPAMT was obtained, and the decay-corrected yield was 34%. The specific activity of this compound was 0.32 Ci/μmol. For the automated synthesis of ¹⁸F-FPAMT, the decay-corrected yield was 15%, the end-of-synthesis time was 110 min, and the specific activity was 0.16 Ci/μmol.

3.3. In Vitro Cellular Uptake Studies

The uptake of ¹⁸F-FPAMT reached saturation at 60 min (Figure 3). ¹⁸F-FDG uptake continued to increase throughout the period, and the percentage uptake of ¹⁸F-FDG was higher than that of ¹⁸F-FPAMT at each time point.

Figure 3: In vitro cellular uptake of ¹⁸F-FPAMT and ¹⁸F-FDG in mesothelioma cells (IL-45). Data are expressed as mean percent of cellular uptake ± standard deviation (%uptake ± SD) measured at 15, 30, 60, and 120 min.

3.4. Biodistribution of ¹⁸F-FPAMT and ¹⁸F-FDG in Mesothelioma-Bearing Rats

The distributions of ¹⁸F-FPAMT and ¹⁸F-FDG in various tissues in mesothelioma-bearing rats are shown in Tables 1 and 2, respectively. Both compounds showed no marked increase in bone uptake, representing their in vivo stability. High kidney and pancreas uptake of ¹⁸F-FPAMT was observed, and this phenomenon was also observed from other tyrosine-based radiotracers [18]. Unlike ¹⁸F-FDG, ¹⁸F-FPAMT had poor uptake in brain tissue.

Table 1: Biodistribution of ¹⁸F-FPAMT in rats.

% of injected dose per gram of tissue weight (/time, interval (iv))

30 min
90 min
180 min

Blood

Heart

Lungs

Thyroid

Pancreas

Liver

Spleen

Kidneys

Stomach

Intestines

Uterus

Muscle

Bone

Brain

Values shown represent the mean ± standard deviation of data from 3 animals.

Table 2: Biodistribution of ¹⁸F-FDG in rats.

% of injected dose per gram of tissue weight (/time, interval (iv))

30 min
90 min
180 min

Blood

Heart

Lungs

Thyroid

Pancreas

Liver

Spleen

Kidneys

Stomach

Intestines

Uterus

Muscle

Bone

Brain

Values shown represent the mean ± standard deviation of data from 3 animals.

3.5. Dosimetry of ¹⁸F-FPAMT in Rats

The estimated absorbed radiation dose of ¹⁸F-FPAMT is shown in Table 3. According to the US Food and Drug Administration Regulations, human exposure to radiation from the use of “radioactive research drugs” should be limited to 3 rem per single administration and 3 rem per year to the whole body, blood-forming organs (red marrow, osteogenic cells, and spleen), the lens of the eye, and gonads (testes and uterus); the limit for other organs is 5 rem per single administration and 15 rem annually. The total rem of ¹⁸F-FPAMT absorbed by each organ was below these limits at the proposed injection of 30 mCi per patient.

Table 3: Radiation dose estimates of reference adult for ¹⁸F-FPAMT.

Target organ
rad/mCi
human dose (mCi)
rad

Organs (5 rem annually/15 rem total)

Adrenals

30
0.089

Brain

30
0.028

Breasts

30
0.059

Gall bladder wall

30
0.086

Lli wall

30
0.095

Small int.

30
0.106

Stomach

30
0.084

Uli wall

30
0.096

Heart wall

30
0.088

Kidneys

30
0.186

Liver

30
0.045

Lungs

30
0.072

Muscle

30
0.049

Pancreas

30
0.101

Bone surfaces

30
0.206

Skin

30
0.047

Testes

30
0.069

Thymus

30
0.073

Thyroid

30
0.074

Urine bladder wall

30
0.090

Uterus

30
0.101

Eff dose

30
0.078

Blood-forming organs (3 rem annually/5 rem total)

Ovaries

30
0.097

Red marrow

30
0.069

Spleen

30
0.103

Eff dose eq.

30
0.094

Total body

30
0.071

3.6. Imaging of Mesothelioma-Bearing Rats

Scintigraphic images of mesothelioma-bearing rats administrated ¹⁸F-FPAMT or ¹⁸F-FDG showed that tumors could be clearly detected, and bone uptake was low (Figure 4). The standardized uptake value (SUV) curve of ¹⁸F-FPAMT for tumor and muscle reached the plateau at 30 min after injection, but the SUV curve of ¹⁸F-FDG for tumor continued increasing during the imaging. The SUV ratios of tumor to muscle for ¹⁸F-FPAMT and ¹⁸F-FDG were 2.82 and 8.26, respectively. There was extremely low uptake of ¹⁸F-FPAMT in the brain and spinal cord when compared with ¹⁸F-FDG (Figure 5).

Figure 4: μPET images of ¹⁸F-FPAMT and ¹⁸F-FDG in mesothelioma-bearing rats (lower body: IL-45, at 45 min). The SUV ratios of tumor to muscle for ¹⁸F-FPAMT and ¹⁸F-FDG were 2.82 and 8.26, respectively. Computer-outlined regions of interest (ROI) (counts per pixel) for tumor and muscle at the corresponding time interval were used to generate a dynamic plot. Dynamic plot was from 0 to 45 minutes.

Figure 5: μPET images of ¹⁸F-FPAMT and ¹⁸F-FDG in mesothelioma-bearing rats (upper body: IL-45, at 45 min). There was extremely low uptake of ¹⁸F-FPAMT in the brain and spinal cord when compared with ¹⁸F-FDG.

4. Discussion

Mesothelioma is an asbestos-related neoplasm generating from mesothelial cells in the pleural, peritoneal, and pericardial cavities, and its incidence increased in several countries [19]. The diagnostic tools and treatment regimens for these tumors are disappointing, and median survival time is 12 months after initial diagnosis [20]. The initial diagnoses of mesothelioma are based on patient’s medical history and physical examination. After that, computed tomography scans and magnetic resonance imaging are used to screen patients, and then biopsy test is needed to confirm the incidence of mesothelioma. ¹⁸F-FDG/PET scan is the tool to determine whether a suspicious area is malignant mesothelioma or a benign condition such as pleural scarring, and the result can identify the best area for an accurate biopsy. PET scans are also effective for highlighting mesothelioma metastases that may not appear on other conventional imaging scans. However, ¹⁸F-FDG/PET scans have limitations in differential diagnosis between cancerous cells and inflammation tissues which metabolize glucose with abnormally high rates. In this case, radiolabeled amino acids are the alternative methods to detect malignant pleural mesothelial and other cancerous cells which overexpress unregulated amino acid transporters [21–23]. Mesothelioma rat model was then selected because rat model provided better anatomical differentiation than mouse model in imaging studies. It is more accurate to determine radiation dosimetry from biodistribution data.

¹⁸F-FET and ¹⁸F-FAMT are radiolabeled amino acids, and they are useful in imaging cancers. However, existing methods for synthesizing these compounds result in low yields, thus limiting the availability of ¹⁸F-FET and ¹⁸F-FAMT in the clinic. In the present study, we synthesized ¹⁸F-FPAMT, an ¹⁸F-FAMT analog, and used a mesothelioma rat model to preliminarily evaluate it as a tumor-imaging compound. We used NMR and mass spectrometry to confirm the structure of ¹⁹F-FPAMT. The yield of ¹⁹F-FPAMT was 46.71%. N-t-butoxycarbonyl-O-[3-tosylpropyl]-α-methyl tyrosine ethyl ester was used as the starting material for manual and automated syntheses of ¹⁸F-FPAMT. The quality control of ¹⁸F-FPAMT was evaluated by radio-TLC and HPLC. Manual synthesis of ¹⁸F-FPAMT resulted in the decay-corrected yield of 34%, radiochemical purity of >95%, the specific activity of 0.32 Ci/μmol, and pH value of 5 to 6; the manual synthesis time was 88 min. Automated synthesis of ¹⁸F-FPAMT resulted in the decay-corrected yield of 15%, radiochemical purity of >95%, the specific activity of 0.16 Ci/μmol, and pH value of 5 to 6; the manual synthesis time was 110 min.

The traditional method of radiosynthesizing ¹⁸F-labeled tyrosine analogs such as ¹⁸F-FET and ¹⁸F-FAMT was through electrophilic substitution reaction which has low synthetic yield. Besides, the reaction uses ¹⁸F-F₂ gas, and HPLC separation makes it even difficult to use this method in automated modules. Although a nucleophilic reaction could result in a high yield of ¹⁸F-FET (40%), this method still requires HPLC for purification, and, thus, it is not ideal to use this synthesis method in automated synthesis modules. In the present study, we obtained ¹⁸F-FPAMT by a nucleophilic reaction, but we completed the purification process without HPLC. Therefore, our method of synthesizing ¹⁸F-FPAMT can be applied to the customized automated synthesis module.

For the in vitro studies, although the result showed that ¹⁸F-FPAMT had lower cellular uptake than that of ¹⁸F-FDG, the uptake mechanism of these two compounds is different. Malignant cells utilize ¹⁸F-FDG as glucose for upregulated aerobic glycolysis and ¹⁸F-FPAMT as an amino acid for proliferation. The results indicate that ¹⁸F-FPAMT has the potential to become a tumor detecting tracer. Biodistribution studies showed that ¹⁸F-FPAMT and ¹⁸F-FDG were rapidly cleared from blood and distributed in other tissues. Compared with ¹⁸F-FDG, the accumulation of ¹⁸F-FPAMT was significantly lower in heart, lungs, thyroid, spleen, and brain. High accumulation of ¹⁸F-FPAMT was observed in the kidneys and pancreas after administration. This could be due to the high expression of the amino acid transporters in the kidneys and pancreas [4]. These results were consistent with those of other radiolabeled amino acid analogs such as ¹⁸F-FAMT [24] and ⁷⁷Br-BAMT [18], although ¹⁸F-FET showed only higher uptake in kidneys [25]. The bone uptakes of ¹⁸F-FPAMT and ¹⁸F-FDG at 180 min after administration increased slightly, suggesting defluorination of both compounds. In the microPET studies of ¹⁸F-FDG and ¹⁸F-FPAMT, the lesions could be observed clearly at 45 min after administration (Figure 4), and the accumulation of ¹⁸F-FPAMT in the brain and spinal cord was significantly less than that of ¹⁸F-FDG (Figure 5), suggesting that ¹⁸F-FPAMT has great potential in imaging brain tumors.

5. Conclusion

In this study, we manually synthesized ¹⁸F-FPAMT with high yielding and radiochemical purity, and we used the customized automated synthesizer for the proof of concept of automated manufacturing of ¹⁸F-FPAMT. Both in vitro and in vivo studies suggested that ¹⁸F-FPAMT can be a good PET agent for detecting mesothelioma, and it might have great potential in brain tumor imaging. In the future, we will focus on optimization of the automated processes for a better yield and a higher specific activity.

Conflict of Interests

All authors have no commercial associations that might pose a conflict of interests in connection with the submitted paper.

Acknowledgments

This paper was supported in part by the John S. Dunn Foundation and the Sponsored Research Agreement (LS01-212) made by Cell > Point L.L.C. at the MD Anderson Cancer Center. The NMR, mass spectrometry, and animal research were supported by the MD Anderson Cancer Center Support Grant.

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